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|Human Cell lysate that Mouse CST3 / Cystatin-C / Cystatin-3 transfected / overexpressed for Western blot (WB) positive control. The whole cell lysate is provided in 1X Sample Buffer (1X modified RIPA buffer+1X SDS loading buffer).|
|A DNA sequence encoding the mouse CST3 (NP_034106.2) precursor (Met 1-Ala 140) was expressed with a C-terminal polyhistidine tag.|
|The secreted recombinant mouse CST3 consists of 131 amino acids and has a calculated molecular mass of 15 kDa. The recombinant protein migrates as three bands (15, 18 & 21-24 kDa) in SDS-PAGE under reducing conditions.|
|Cell lysate was prepared by homogenization in ice-cold modified RIPA Lysis Buffer with cocktail of protease inhibitors (Sigma). Cell debris was removed by centrifugation. Protein concentration was determined by Bradford assay (Bio-Rad protein assay, Microplate Standard assay). The cell lysate was boiled for 5 min in 1 x SDS loading buffer (50 mM Tris-HCl pH 6.8, 12.5% glycerol, 1% sodium dodecylsulfate, 0.01% bromophenol blue) containing 5% b-mercaptoethanol, and lyophilized.|
|Modified RIPA Lysis Buffer: 50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 1% Sodium deoxycholate, 1mM PMSF.|
|12.5% SDS-PAGE Stained with Coomassie Blue after protein purification.|
|Samples are stable for up to twelve months from date of receipt.|
|1. Centrifuge the tube for a few seconds and ensure the pellet at the bottom of the tube. 2. Re-dissolve the pellet using 200μL pure water and boil for 2-5 min. 3. Store the lyophilized cell lysate at 4℃. After re-dissolution, recommend to aliquot it into smaller quantities and store at -80℃.|
|1 X Sample Buffer (1 X modified RIPA buffer+1 X SDS loading buffer).|
|Store at 4℃. After re-dissolution, aliquot and store at -80℃.|
|Western blot (WB): Use at an assay dependent dilution.|
Other Applications: Not tested.
Optimal dilutions/concentrations should be determined by the end user.
Cystatin C, also known as Cystatin-3 (CST3) is a secreted type 2 cysteine protease inhibitor synthesized in all nucleated cells, has been proposed as a replacement for serum creatinine for the assessment of renal function, particularly to detect small reductions in glomerular filtration rate. The mature, active form of human cystatin C is a single non-glycosylated polypeptide chain consisting of 120 amino acid residues, with a molecular mass of 13,343-13,359 Da, and containing four characteristic disulfide-paired cysteine residues. Cystatin C is a low-molecular-weight protein which has been proposed as a marker of renal function that could replace creatinine. Indeed, the concentration of Cystatin C is mainly determined by glomerular filtration and is particularly of interest in clinical settings where the relationship between creatinine production and muscle mass impairs the clinical performance of creatinine. Since the last decade, numerous studies have evaluated its potential use in measuring renal function in various populations. More recently, other potential developments for its clinical use have emerged. In almost all the clinical studies, Cystatin C demonstrated a better diagnostic accuracy than serum creatinine in discriminating normal from impaired kidney function, but controversial results have been obtained by comparing this protein with other indices of kidney disease, especially serum creatinine-based equations, such as early atherosclerosis, Alzheimer's dementia, vascular aneurysms, hyperhomocysteinaemia and other neurodegenerative diseases. Cystatin C could be a useful clinical tool to identify HIV-infected persons. In addition, its expression is up-regulated in malignance of certain tumor progression.